T1DM results from the immune-mediated destruction of the body's only insulin producing cells, the pancreatic beta cells. Beta cells comprise only about 2% of the pancreatic cell number and are grouped together into cell clusters with alpha cells (making glucagon), delta cells (making somatostatin), F cells (making pancreatic polypeptide), and other rare cells to form "mini-organs" called islets of Langerhans, or simply "islets." Isolated destruction of islet alpha, delta, or F cells has not been described. In contrast, isolated immune-mediated beta cell destruction does occur such that pancreata from T1DM animal models or from patients with newly diagnosed T1DM reveal "islet remnants", i.e. small islets with few or no beta cells, but apparently normal numbers of the other islet cell types. Over time however, clinical evidence indicates that patients with T1DM also lose alpha cell function suggesting that normal alpha cells are dependent upon normal beta cell function. The "mini-organ" concept is apt for at least 2 other reasons: (1) the different islet cell types are organized in a typical pattern: beta cells centrally located and the other cells types located more peripherally, and (2) islets consume more pancreatic blood flow (about 20%) than their small mass would suggest. Thus islets are important, not well-understood structures with unique physiological and immunological features, and they represent an Achilles' heel for individuals destined to develop T1DM. This project has seven parts, to: (1) improve isolation techniques to more predictably yield high quality islets, (2) improve assays for characterizing isolated islet quality, (3) test, using a non-human primate islet transplant model, important pre-clinical questions, (4) perform human clinical islet transplants under carefully planned protocols, (5) develop assays for characterizing islet function post-transplant, and (6) develop a renewable islet source, and (7) test novel ways of preventing islet allograft rejection following transplant. Beginning in 7/99, in collaboration with the Clinical Center's Department of Transfusion Medicine/Cell Processing Unit and Dr. Ricordi (University of Miami's Diabetes Research Institute), islets have been isolated from 65 human pancreata and since 9/00, from 17 rhesus monkeys. Before initiating the isolations, we established two separate laboratories, one for human glands and one for animals, and we trained a cadre of technicians. State of the art islet isolation units typically achieve islet yields of only about half the pancreatic total, and even that level is achieved about half the time. The NIH team matches that islet yield standard but is testing ways to improve it. Currently available assays testing islet viability and function in vitro do not correlate with the imperfect but gold standard assay for in vivo function, i.e. islets transplanted into diabetic NOD-scid mice. We have established the capability to perform the standard in vitro islet function assays (islet insulin release in low- and high-glucose media, and viability assays) and the in vivo NOD-scid transplant model. Due to the special expertise required for the latter, we now typically send islets to collaborators at Vanderbilt University (Dr. Alvin Powers) for the NOD-scid assays. We also initiated plans to study new in vitro assays including efforts to study islets using microarray techniques, and the regulation of insulin biosynthesis at the translational level. We initiated rhesus monkey experiments. We studied isolated monkey islets in vitro and transplanted islets into diabetic rhesus monkey recipients. The rhesus monkey islet transplant model allowed us to demonstrate that: (1) that NIH-isolated islets were viable and functional, (2) the portal vein infusion site is superior to intra-arterial infusion (manuscript submitted) and (3) diabetes can be effectively and more safely induced (compared with the more typically employed surgical pancreatectomy approach) via intra-arterial infusion of a beta cell toxin call streptozotocin. The model will not be utilized to test new questions like the optimal site for islet infusion, newer anti-rejection therapies, detailed metabolic studies before and after islet transplantation, and whether islets can be induced to differentiate from progenitor cells injected into the pancreas. We are testing ways to improve on the difficult and expensive primate model. One, we are establishing collaboration with University of Maryland investigators who maintain a colony of spontaneously diabetic rhesus monkeys. Two, we collaborate with a Yale University interventional radiologist to find less invasive ways of transplanting the islets. All these studies are designed to support islet transplant clinical trials at the NIH. In the past year, we have transplanted islets into 6 individuals with T1DM of at least 5 years duration, and with a clinical evidence of "brittle" disease. All patients display clear evidence that their transplanted islets continue to function in that 3 are insulin independent with normal Hgb A1c values, and 3 others are on 50% or less of their pre-transplant insulin dose, and with improved blood glucose control and no serious hypoglycemia. We, like others, believe these data demonstrate that islets transplanted into patients with long-standing T1DM can improve quality of life by alleviating or eliminating the need for exogenous insulin. Unfortunately, we also recognize that before islet cell transplantation can be considered a widely applicable therapy a renewable islet source will need be identified, and better way to prevent islet graft failure need be tested and perfected. We have therefore initiated studies to test whether pancreatic stem cells can be identified and/or stimulated to proliferate (collaboration with Dr. Nadya Lumelsky), ways to non-invasively measure islet mass in vivo (Dr. Anthony Basile), and novel ways to prevent islet allograft rejection.